Antigen

Antigen Class

TFs and others

Antigen

Ascl1

Cell type

Cell type Class

Neural

Cell type

Basal Ganglia

MeSH Description

Large subcortical nuclear masses derived from the telencephalon and located in the basal regions of the cerebral hemispheres.

Sample

source_name

brain

tissue

brain

cell line

basal ganglia

cell type

E13.5

genotype

wild-type

chip antibody

BD, cat# 556604

geo_loc_name

missing

collection_date

missing

Sequenced DNA Library

library_name

GSM6917350

library_strategy

ChIP-Seq

library_source

GENOMIC

library_selection

ChIP

library_construction_protocol

ChIP was performed using antibodies against SP9, ARX, ASCL1, GSX2, and NR2F1. Basal ganglia were dissected in cold PBS from CD1 embryos (2 L/Ab for NR2F1; 3 L/Ab for ARX, ASCL1, and GSX2). The basal ganglia consisted of the LGE, MGE and CGE progenitor and mantle zones. The dissected basal ganglia were either fixed in 1% formaldehyde at RT for 10 min (SP9, ASCL1, GSX2, NR2F1) or fixed in 1.5% formaldehyde at RT for 20 min (ARX), neutralized with glycine, and washed gently in PBS. The fixed cells were lysed with a hypotonic buffer (50 mM Tris pH 7.5 / 0.5% NP40 / 0.25% sodium deoxycholate / 0.1% SDS / 150 mM NaCl) to obtain the nuclei; these were then lysed in 1% SDS buffer and the chromatin was sheared into 300-1000 bp fragments by sonicating for 40 cycles (30 sec on and 45 sec off) using a bioruptor (Diagenode). Immunoprecipitation (IP) reactions were performed with the sheared chromatin diluted 1/10 times with “dilution buffer” (0.01% SDS, 1.1% Triton X- 100, 1.2 mM EDTA, 16.7 mM Tris-HCl, pH 8.1, 167mM NaCl, usually in 6 ml. Antibody was then added to either 5 µg (ARX, SP9, NR2F1) or 8 µg (ACSL1, GSX2) specific antibodies. Negative control ChIP reactions used either IgG (5µg) or blocking peptide (ARX, 400x molar excess). Antibody/chromatin complexes were purified using Dynabeads (Invitrogen) and washed extensively in “wash buffer” (low salt, 0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, pH 8.1, 150 mM NaCl; high salt, 0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, pH 8.1, 500 mM NaCl; LiCl, 0.25 M LiCl, 1% IGEPAL CA630, 1% deoxycholic acid (sodium salt), 1mM EDTA, 10mM Tris, pH 8.1 and TE). Complexes were eluted with 1% SDS, 10 mM sodium bicarbonate buffer at 65 ºC for 10 min. Eluted chromatin was reverse-crosslinked overnight at 65 ºC in the presence of 500 mM NaCl, then subsequently treated with RNase (10 µg/200 µL reaction, 15 min at 37 ºC) and Proteinase K (10 µg/200 µL reaction, 60 min at 55 ºC) and cleaned using a ChIP DNA Clean & Concentrator kit (Zymo Research). The chromatin was quality-controlled (QC) using qPCR to check for enrichment of genomic DNA fragments that were expected, and not expected, to have the different TF binding. For ChIP-seq, libraries were prepared using an Ovation Ultralow DR Multiplex System (Nugen), size- selected in the range of 300 bp on a chip from BluePippin (Sage Science) and lastly QC tested on a Bioanalyzer (Agilent). The libraries were sequenced as single-end 50-bp reads on a HiSeq 4000 (Illumina) at the Center for Advanced Technology (UCSF). PLAC-seq libraries for E13.5 basal ganglia were prepared similar to a previously published protocol61. 3 to 7 million cells were used for each library. If the cells appeared aggregated, they were dissociated with gentle MACS dissociator or Dounce homogenizer. Each PLAC-seq library was prepared using DpnII as the restriction enzyme and Dynabeads M-280 sheep anti-rabbit IgG (Invitrogen #11203D) mixed with 5 µg of H3K4me3 (04-745, Millipore) for the chromatin immunoprecipitation step. Finally, libraries were prepared with the Illumina TruSeq adaptors and the final libraries were sent for paired-end sequencing on the HiSeq X Ten (150-bp reads) equipment. ChIP-seq, PLAC-seq

Sequencing Platform

instrument_model

Illumina HiSeq 4000

mm10

Number of total reads

30334626

Reads aligned (%)

97.3

Duplicates removed (%)

12.0

Number of peaks

414 (qval < 1E-05)

mm9

Number of total reads

30334626

Reads aligned (%)

97.0

Duplicates removed (%)

12.0

Number of peaks

495 (qval < 1E-05)